It is well established that live attenuated micro-organisms can be highly effective vaccines; immune responses elicited by such vaccines are often of greater magnitude and of longer duration than those produced by non-replicating immunogens. One explanation for this may be that live attenuated strains establish limited infections in the host and mimic the early stages of natural infection. In addition, unlike killed or inactivated preparations, live vaccines are able to induce potent cell-mediated responses which may be connected with their ability to replicate in antigen-presenting cells, such as macrophages.
There has been a long history of the use of live attenuated vaccines in animals and humans, notably using chemical mutagenesis techniques. However, empirically attenuated vaccines can revert to virulence.
Modem molecular biology techniques, coupled with the increasing knowledge of bacterial pathogenesis, has led to the identification of several genes that are involved in the growth and survival of the micro-organisms in vivo. This has provided new gene targets for attenuation, and to the concept that future vaccine strains could be ‘rationally’ attenuated by introducing defined non-reverting mutations into selected genes known to be involved in virulence, see for example WO-A-00/61724, WO-A-00/68261 and EP-A-0889120.
Although many attenuated strains have been produced in laboratories, only a few have qualified as potential vaccine candidates for use in animals. This may be due in part to the need to balance the immunogenicity of the vaccine with the possibility of the micro-organism to revert, becoming reactive and pathogenic.
It is clear that the selection of appropriate genes for attenuation, which will result in a suitable vaccine candidate, is not straightforward and cannot easily be predicted. Many factors may influence the acceptability of an attenuated mutant as a vaccine, and consequently research effort is required to identify and select suitable attenuating genes. Many attenuation experiments were conducted only in vitro and their results cannot be extrapolated in vivo, notably in relation to residual pathogenicity of the resulting mutants for the vaccinated animals.
Mention is made of:                Kachlany S C, Planet P J, Bhattacharjee M K, Kollia E, DeSalle R, Fine D H, Figurski D H., Nonspecific adherence by Actinobacillus actinomycetemcomitans requires genes widespread in bacteria and archaea. J Bacteriol. 2000 November; 182(21):6169-76.        Fuller T E, Martin S, Teel J F, Alaniz G R, Kennedy M J, Lowery D E., Identification of Actinobacillus pleuropneumoniae virulence genes using signature-tagged mutagenesis in a swine infection model. Microb Pathog. 2000 July; 29(1):39-51.        Fuller T E, Kennedy M J, Lowery D E., Identification of Pasteurella multocida virulence genes in a septicemic mouse model using signature-tagged mutagenesis. Microb Pathog. 2000 July; 29(1):25-38.        Kehrenberg C, Werckenthin C, Schwarz S., Tn5706, a transposon-like element from Pasteurella multocida mediating tetracycline resistance. Antimicrob Agents Chemother. 1998 August; 42(8):2116-8.        DeAngelis P L., Transposon Tn916 insertional mutagenesis of Pasteurella multocida and direct sequencing of disruption site. Microb Pathog. 1998a April; 24(4):203-9.        DeAngelis P L, Jing W, Drake R R, Achyuthan A M., Identification and molecular cloning of a unique hyaluronan synthase from Pasteurella multocida. J Biol Chem. 1998b April 3; 273(14):8454-8.        Lee M D, Henk A D., Tn10 insertional mutagenesis in Pasteurella multocida. Vet Microbiol. 1996 May; 50(1-2):143-8.        Choi K H, Maheswaran S K, Choi C S., Colorimetric assay using XTT for assessing virulence of avian Pasteurella multocida strains. Vet Microbiol. 1995 July; 45(2-3):191-200.        Nnalue NA. Tn7 inserts in both orientations at a single chromosomal location and apparently forms cointegrates in Pasteurella multocida. Mol Microbiol. 1990 January; 4(1):107-17.        Stocker U.S. Pat. Nos. 4,550,081, 4,837,151, 5,210,035 and 5,643,771.        Highlander U.S. Pat. No. 6,180,112.        
Kachlany involved Tad genes. There is no relation between the Tad genes mutated in Kachlany and attenuation. There is no testing on animals in Kachlany and the Tad genes are not selected in the present invention. The Fuller papers involve sequences that are not selected in the present invention. Kehrenberg did not involve an attenuated mutant, or a Signature Tagged Mutagenesis or STM technique; but rather, Kehrenberg involved a directed insertion of a transposon (use of identical insertion element). DeAngelis 1998a provides only a general description of a STM technique, and nothing about mutants, per se. DeAngelis 1998b involved the use of a STM technique to insert a transposon in the HA biosynthesis locus (Genbank AF036004). This sequence is a homologue to the sequence Pm0775 of PM70. The sequence encoding Pm0775 is not selected in the present invention. Lee concerns the use of a STM technique with a Tn10 transposon; Lee fails to disclose or suggest any tests on animals or any searches for attenuated mutants; but rather, Lee involved only auxotrophic mutants. While Choi cites a Pasteurella multocida transposon insertion mutant, and there may have been no mortality induced by this mutant, Choi contains no details about the location of the transposon insertion and therefore cannot be said to be reproducible. Nnalue similarly fails to teach or suggest the instant invention. The Stocker patents involved the insertion of a Tn10 transposon in the aroA gene. AroA gene is not selected in the present invention. Highlander concerns the insertion of a Tn1545 transposon in the lktC gene to inactive leukotoxin. LktC gene is not selected in the instant invention. Accordingly, it is verily believed that the instant invention is not taught or suggested in the art.
Moreover, it is desirable to characterize genes or nucleic acid sequences involved in attenuation and on this basis develop attenuated bacteria, as well as attenuated vaccines or immunogenic compositions, such as those having a high degree of immunogenicity and which exhibit a good safety profile with limited or no side effects.